Figure 3

Bead halo assay. (a–d) An example of an NTR–cargo pair that does not bind. (e–h) An example of an NTR–cargo pair that does bind. (a) A GST-mCherry-NTR is fixed on GSH-Sepharose beads. If GFP-cargo does not bind to the NTR, it remains in the buffer around the beads. (e) If cargo binds to the NTR, it is concentrated on the bead surface. (b, f) Differential interference contrast (DIC) microscopic images of the beads. (c, g) Confocal microscopic images of mCherry fluorescence. The yellow square in (g) indicates the magnified region in (j, k). (d, h) Confocal images of GFP fluorescence. (i) After background subtraction, the GFP/mCherry fluorescence ratio around the beads was used as the index for the binding intensity. ROI and BG were defined as in (j, k). The region mean intensities in 12-bit images were used for the calculation. (j) The ROI was set on the mCherry fluorescent image as ring-shaped regions with a 5 pixel width along the inside of the outlines of beads (yellow double line). The same ROI was applied to the corresponding GFP fluorescent image. (k) The BG was established as larger ring-shaped regions with a 5 pixel width and 5 pixels away outside the ROI. Images in (j, k) are magnifications of the square region in (g). In the actual quantitation, ROIs and BGs were set on original images with dimensions of 1024 × 1024 pixels, of which the field size (317 × 317 µm) was the same as that of the images (b–d and f–h). Scale bar: 100 µm. For more examples of the ROI setting, see Supplementary Fig. S2.